CN109563056B

CN109563056B - Triazoles for modulating intracellular calcium homeostasis

Info

Publication number: CN109563056B
Application number: CN201780045689.1A
Authority: CN
Inventors: A·瓦列霍伊拉尔塔曼迪; A·J·罗帕斯德穆纳安阿瑞格; P·费隆塞尔马; J·M·艾兹普鲁伊帕拉古里; A·伊拉斯托萨埃佩尔德; J·I·米兰达穆鲁亚; I·泰勒奥杰达; G·阿尔达农多阿里斯蒂萨瓦尔
Original assignee: University Pais Vasco; Administracion General de la Comunidad Autonoma de Euskadi
Current assignee: University Pais Vasco; Administracion General de la Comunidad Autonoma de Euskadi
Priority date: 2016-05-24
Filing date: 2017-05-23
Publication date: 2022-08-16
Anticipated expiration: 2037-05-23
Also published as: JP2019520423A; CA3025436A1; RU2018141584A; DK3466933T3; RU2753509C2; HUE053371T2; CA3025436C; PT3466933T; IL263168B2; ES2643856A1; RU2018141584A3; CL2018003318A1; CN109563056A; JP6983875B2; ES2643856B1; IL263168B1; US11377427B2; AU2017270485B2; WO2017203083A1; HRP20210211T1

Abstract

The invention relates to 1,2, 3-triazoles of formula (I):

Description

Triazoles for modulating intracellular calcium homeostasis

Technical Field

The present invention relates to novel substituted 1,2, 3-triazoles useful for increasing or restoring intracellular calcium homeostasis in human and animal cells. The invention also relates to methods for the synthesis of said compounds, pharmaceutical compositions comprising the same, and the use thereof for the prevention or treatment of skeletal muscle, cardiac and neurodegenerative disorders.

Background

Muscular dystrophy is a heterogeneously inherited disease characterized by progressive weakness and atrophy of skeletal muscles. With X Duchenne Muscular Dystrophy (DMD) being one of the most common forms, 1 in 3500 males suffers. As in the case of its more benign allelic form (becker muscular dystrophy, BMD), DMD and BMD are both caused by mutations in the gene encoding the dystrophin (dystrophin), a 427-kDa cytoskeletal protein. Because of the high incidence of sporadic cases, genetic studies are not sufficient to eradicate the disease, and there is an urgent need for the development of new effective therapies.

Limb Girdle Muscular Dystrophy (LGMD) is a large group of inherited muscular dystrophies characterized by progressive proximal weakness, mainly involving the pelvic girdle and shoulder girdle. Among the recessive forms of LGMD, calpain (calpainpopathy) or LGMD2A are the most common forms and are caused by mutations in the gene encoding calpain 3(CAPN3), a non-lysosomal cysteine protease required for normal muscle functioning and regeneration.

Myotonic dystrophy type 1 (DM1) is the most common form of adult muscular dystrophy and is characterized by muscle weakness, myotonia and multiple system involvement. It is a dominant autosomal inherited disease caused by the unstable expansion of the CTG triplet repeat in the 3' noncoding region of the DMPK gene, located on the long arm of chromosome 19and expressed mainly in skeletal muscle.

These and other muscular dystrophies (e.g., DM2, recessive and dominant LGMD, congenital or metabolic myopathy, etc.) all have altered intracellular calcium homeostasis. Due to the fact that intracellular Ca is present in the muscle fibers ²⁺ Changes in concentration appear to represent a central universal pathogenesis, preventing intracellular Ca ²⁺ The development of altered therapeutic interventions is a very valuable therapeutic goal. (Vallejo-Illarramendi et al. expert Rev. molecular. Med.2014,16, e16, doi: 10.1017/erm 2014.17 "Dysrelation of calcium hoscostasis in mammalian dynamics").

High baseline intracellular Ca ²⁺ The levels result in calpain activation, protein degradation, opening of the mitochondrial penetrating transition pore (mPTP), and ultimately death of muscle fibers due to necrosis. Intracellular Ca ²⁺ The level increase is related to Ca passing through the sarcolemma ²⁺ Flux (flux), calcium loss from Sarcoplasmic Reticulum (SR) and Ca in SR ²⁺ A complex process of horizontal anomalies. In mdx mice, an animal model of duchenne muscular dystrophy, aberrant S-nitrosylation of cysteine residues of srylandine cinnamon salt (ryanodine) receptors RyR1 and RyR2 leads to dissociation of calstabin (calstabin) from the protein complex, which creates an unstable channel of calcium loss at rest. This nitrosation appears to be caused by an abnormal regulation of nitric oxide, which causes nitrosation and oxidative stress in the muscle of dystrophic mdx mice (Dudley et al, am. j. pathol.2006,168, 1276-1284; Bellinger et al, Nat med.2009,15, 325-. Furthermore, expression of micromodulin caused in dystrophin-deficient microtubules restored the increased myo-inositol 1,4, 5-triphosphate receptor (IP3R) -dependent calcium release to control levels, indicating that IP3R is involved in Ca in DMD ²⁺ The steady state changes.

By immunoprecipitation experiments, it was demonstrated that calpain 3(CAPN3) interacts with Cryptocalcitonin (CSQ), a protein involved in calcium homeostasis. In CAPN3 knockout mice (C3KO, Capn3-/-), reduction in RyR1 levels was accompanied by Ca ²⁺ The release from the SR decreases. Furthermore, it has been found that Ca is released after calcium release from SR in Capn 3-/-mice ²⁺ Reuptake in SR occurs more slowly, although the basis of this finding requires more intensive research. However, it has been observed that loss of CAPN3 proteolytic activity in Capn3cs/cs mice does not affect calcium homeostasis, suggesting that the structural function of CAPN3 is to maintain Ca homeostasis ²⁺ The key to the steady state.

Myotonic dystrophy is associated with dysregulation of alternative (alternative) junctions of the CACNA1S gene encoding the α 1S subunit of the dihydropyridine receptor DHPR, an important voltage sensor in excitation-contraction (E-C) coupling. In DM1 and DM2, there was a large CACNA1S exon 29 omission (deletion), which increased channel conduction and voltage sensitivity.

Several disorders and diseases are associated with congenital or acquired modifications of the RyR1 protein (Kushmir et al, recent patbotbiotechnol.2012, 6,157-166 "Ryanodine receptor patents"). Such disorders and diseases include skeletal muscle, cardiac and nervous system conditions. More specifically, it includes, but is not limited to, congenital myopathy, muscular dystrophy, sarcopenia, skeletal muscle fatigue, acquired muscle weakness or atrophy, malignant hyperthermia, exercise-induced cardiac arrhythmia, congestive heart failure, hypertrophic cardiomyopathy, alzheimer's disease, and age-related memory loss. All of these have been proposed to increase intracellular Ca at rest (suppressing conditions) ²⁺ Concentration ([ Ca ] ²⁺ ] _i ) All directly contribute to the toxicity of the cells (myofibers, cardiomyocytes, neurons or glial cells), their deterioration and Ca ²⁺ Simultaneous activation of dependent proteases.

There are two main types of calcium channel-associated srilanca cinnamomi receptors in muscle fibers: RyR1 located in skeletal muscle and RyR2 located in cardiac muscle. Each calcium channel is formed by a tetramer of RyR proteins, each of which is capable of interacting with calponin. RyR1 binds to FKBP12 (calpain 1), whereas RyR2 binds to FKBP12.6 (calponin 2). It has been found in both cardiac and skeletal muscle that abnormal dissociation of calponin from RyR channels, caused by progressive nitrosylation of RyR channels, leads to Ca from the sarcoplasmic reticulum to the intracellular cytoplasm ²⁺ Increased release, decreased muscle performance during contraction and long-term activation of muscle dysfunction (Bellinger et al, nat. Med.2009,15, 325-.

Some low molecular weight compounds that show therapeutic activity in damaged muscle tissue are known in the prior art. For example, 4- [3- (4-benzylpiperidinyl) -propionyl has been demonstrated]-2,3,4,5-tetrahydro-1,4-benzothiazepine

(JTV-519) for use in the prevention of myocardial necrosis and myocardial infarction. At 10 ^–6 M concentration, compound JTV-519 inhibited epinephrine-and caffeine-induced myocardial necrosis in the left ventricle of rat heart in vitro without affecting the heart rate or pressure of the left ventricle (Kaneko, N.et al. WO92/12148A1 "prediction of4- [ (4-benzylpiperidine) -alkanoyl)]-2,3,4,5-tetrahydro-1,4-benzothiazepine derivatives for deactivating the said kinetic cell depletion of cardiac muscles with deactivating cardiac functions "). The compound JTV-519 acts on the calpain 2-associated RyR2 srilankan cinnamon salt receptor (FKBP12.6), increasing the affinity of FKBP12.6 for PKA kinase phosphorylation and for the mutant RyR2 receptor RyR2 receptor, otherwise the mutant RyR2 receptor has reduced affinity for FKBP12.6 or does not bind to FKBP 12.6. This action of JTV-519 repairs Ca in RyR2 ²⁺ Ion leakage (Marks, A.R. et al. US2004/229781A1 "Compounds and methods for treating and preserving exsicase-induced cardiac arrhythmias").

It is also known to include the compound S-107 or other structurally related tetrahydrobenzothiazines

The pharmaceutically active composition of (1) is useful for treating or preventing disorders or diseases associated with RyR2 receptors that regulate the operation of calcium channels in heart cells (Marks, A.R. et al. US2006/194767A1 "Benzothiazepines and novel agents for the prevention and treatment of disorders involving the accumulation of the RyR receptors and the preparation and pharmaceutical compositions", see also Mei, Y.et al. PLoS one.2013,8: e54208 "Stabilization of the same polypeptide expression channel-FKBP12complex the1,4-benzothiazepine derivative S107"). The activity of the family of compounds as RyR 1-calpain 1 interaction stabilizers, reduction of muscle fatigue (Marks, a.r. et al, wo2008/060332a2 "Methods using tetrahydrobenzothiazepine compounds for treating or reducing muscle fatigue"), and for the treatment of sarcopenia (Marks, a.r. et al, wo2012/019071a1 "Methods and compositions for treating deforming and treating sarcopenia") are also known.

Several carvedilol (carvedilol) derivatives have been described that assist in the normalization of intracellular calcium homeostasis by acting on the RyR2 receptor, thereby providing beneficial effects in cardiac therapy (Chen, s.et al. us2007/025489a1 "Preparation of carbazoles as a ryanodine receptor type 2(RyR2) antagonists for treatment of cardiac conditions").

Finally, the sryR 3 receptor isoform of the Srilanca cinnamon salt is known to regulate intracellular calcium homeostasis in the brain or other neural tissues, and the use of benzothiazepines has been advocated

The modulation of which is used to treat certain neuronal disorders (Marks, a.r. et al, wo2012/037105a1 "Methods and compositions for treating or preventing disorders-induced neuronal disorders and diseases"). Furthermore, both RyR1 and RyR2 are expressed in the brain and there is evidence that various brain disorders and neuronal death are involved in calcium regulation and nitrooxidative stress (Kakizawa et al, EMBO J.2012,31,417-50,1055-1067)。

These prior art documents all clearly show that the development of compounds that allow modulation or modulation of RyR receptors by modulating intracellular calcium levels would provide a useful alternative for the treatment of muscular disorders as well as cardiac and neurodegenerative diseases.

Disclosure of Invention

The authors of the present invention have developed novel triazole derivative compounds, in particular, 4- [ (phenylthio) alkyl ] -1H-1,2, 3-triazole, which are suitable for modulating RyR receptors that modulate calcium function in animal or human cells. The compound is referred to herein as "AHK".

As is clear from the experimental section, the compounds according to the invention have the ability to modulate intracellular calcium homeostasis in dystrophic muscle fibers, allowing the increase in intracellular calcium observed to be restored. Furthermore, the compounds have a modulating effect on RyR, which has been demonstrated for the ability to restore RyR 1-calponin interactions on the myotubes of healthy humans experiencing nitrooxidative stress.

Furthermore, in vivo experiments have clearly shown that said compounds additionally allow to increase the grip strength of dystrophic mice, as well as to normalize overexpressed dystrophin genes and to reduce dystrophic histopathological markers.

Accordingly, a first aspect of the present invention relates to 1, 4-disubstituted 1,2, 3-triazole compounds of formula (I):

wherein:

R ¹ is optionally selected from C _1-4 Alkyl radical, C _6-10 Aryl, F, Cl, CN and NO ₂ C substituted by one or more substituents of ₁ -C ₄ An alkylene diradical;

R ² is C ₁ -C ₆ Alkylene diradicals of which 1,2 or 3 are-CH ₂ -the group can optionally be replaced by a group selected from-O-and-S-; and wherein C ₁ -C ₆ The alkylene diradicals can be optionallyIndependently selected from C _1-4 Alkyl, allyl, propargyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 3-aminopropyl, 4-aminobutyl, 3-guanidinopropyl, 3-indolylmethyl, C _6-10 Aryl, benzyl, 4-hydroxybenzyl, C _6-10 Heteroaryl, F, Cl, OH, O (C) _1-4 Alkyl), CN, NO ₂ 、CO(C _1-4 Alkyl), CO ₂ (C _1-4 Alkyl), -CONH (C) _1-4 Alkyl), -CON (C) _1-4 Alkyl radical) ₂ Substituted with one or more groups of (a);

R ³ is independently selected from H, C _1-4 Alkyl radical, C _6-10 Aryl, F, Cl, Br, I;

m is selected from 0,1, 2,3 and 4;

n is selected from 0,1 and 2;

x is independently selected from OH, O (C) _1-4 Alkyl), O (C) _6-10 Aryl), OCF ₃ 、S(C _1-4 Alkyl), S (C) _6-10 Aryl group), C _1-6 Alkyl, CF ₃ 、NHC(O)(C _1-4 Alkyl) and halogen; or two X groups may represent methylenedioxy, ethylenedioxy or propylenedioxy diradicals; and

y is selected from-OH and-CO ₂ H、-CO ₂ (C _1-4 Alkyl), -CO ₂ (allyl), -CO ₂ (benzyl), -SO ₃ H、-NH ₂ 、-NH(C _1-4 Alkyl), -N (C) _1-4 Alkyl radical) ₂ 、N(C _1-4 Alkyl radical) ₃ and-N (heterocyclyl or heteroaryl), wherein said heterocyclyl or heteroaryl is optionally substituted with C _1-4 Alkyl substituted, and wherein the N atom is part of a heterocyclyl or heteroaryl group;

or a pharmaceutically acceptable stereoisomer, salt, solvate or complex thereof, or an isotopically-labeled derivative or prodrug thereof.

Another aspect of the invention includes a method for synthesizing a compound of formula (I), comprising:

a) reacting an alkyne of formula (II) with an azide of formula (III), optionally in the presence of a copper catalyst and optionally in the presence of a base,

to produce a compound of formula (I) as hereinbefore defined,

wherein:

the radical R in the compounds of the formulae (II) to (III) ¹ 、R ² 、R ³ M, n and X are as defined above, and

y is a group as defined above, optionally protected with a carboxyl protecting group, a hydroxyl protecting group or an amino protecting group;

b) when n is 0 in the compound of formula (I) obtained in step a), optionally treating said compound of formula (I) with an oxidizing agent to yield a compound of formula (I) as follows: wherein n is 1 or 2, R ¹ 、R ² 、R ³ M and X are as defined above, and Y is a group as defined above, optionally protected with a carboxyl protecting group, a hydroxyl protecting group or an amino protecting group; and

c) when the compound of formula (I) obtained in step a) or b) has a Y group protected with a protecting group, removing the protecting group to yield a compound of formula (I): wherein R is ¹ 、R ² 、R ³ M, n, X and Y are as defined above.

Another aspect of the invention relates to a pharmaceutical composition comprising a compound of formula (I) as defined above, together with one or more pharmaceutically acceptable excipients or carriers.

Another aspect of the invention relates to the use of a compound of formula (I) as defined above for the preparation of a pharmaceutical product.

A final aspect of the present invention relates to the use of a compound of formula (I) as defined above for the preparation of a pharmaceutical product for the treatment and/or prevention of disorders or diseases associated with dysregulation of intracellular calcium concentration or dysfunction of RyR receptors, in particular, skeletal muscle disorders or diseases, cardiac disorders or diseases and neurological disorders or diseases.

Drawings

FIG. 1 shows a toxicity curve showing the toxicity of compounds AHK1, AHK2 and S-107 to human myotubes after 24 hours incubation using the CytoTox 96 colorimetric assay.

Figure 2 shows the in vitro effect of compounds AHK1 and AHK2 on intracellular calcium levels in mouse muscle fibers at rest:

A) images of fibers isolated from the flexor digitorum brevis showed a characteristic striped pattern. B) Representative images of fibers [ control fibers (CTRL) and dystrophic fibers (MDX) ] loaded with fura-2AM after performing the background corrections required to measure intracellular calcium levels. C) Histograms showing baseline intracellular calcium levels in different groups of fibers. The number of fibres analyzed (n) is shown in the histogram (Kruskal-Wallis and U Mann-Whitney,. p < 0.05).

Figure 3 shows the in vitro effect of compounds AHK1 and AHK2 on RyR 1-calponin 1 interaction in healthy human myotube cultures exposed to peroxynitrite stress.

Figure 4 shows the effect of grip strength on dystrophic mdx mice treated with compounds AHK1 and AHK 2. P < 0.05; n-10 control mice (CTRL); n-11 untreated MDX Mice (MDX); n-10 MDX mice treated with AHK1(MDX + AHK 1); and n-6 MDX mice treated with AHK2(MDX + AHK 2).

Figure 5 shows the in vivo effect of compounds AHK1 and AHK2 on muscle degeneration/regeneration in dystrophic mdx mice:

A) representative frozen sections of control and dystrophic mouse septa were observed in which collagen IV labeled with fluorescent antibodies and nuclei labeled with 4', 6-diamidino-2-phenylindole (DAPI) were observed.

B) Number of central nuclei obtained in sections of control mice (CTRL), dystrophy Mice (MDX) and dystrophy mice treated with AHK1(MDX + AHK1) and AHK2(MDX + AHK 2). P < 0.05; n-3 CTRL; n is 7MDX ND; n-7 MDX + AHK1 and n-4 MDX + AHK 2).

Figure 6 shows the effect of AHK1 treatment on the gene expression pattern of tibialis anterior in mdx mice.

Figure 7 shows the in vivo effect of compound AHK2 on abnormal CNS effects (functionalization) in dystrophic mdx mice:

A) 1 minute traces of control mice (Ctl), mdx mice (mdx), and mdx mice treated with AHK2(mdx AHK2) after a brief 15 second fixation;

B) the increased defense response after acute stress is expressed as the percentage of time that a mouse exhibits immobility (quiescence) over a1 minute duration. (gray column): a tonic immobility time of at least 1 second, wherein the immobility sensitivity is 90%; (black column): percentage of immobility time for non-immobilized animals.

P <0.05 (unpaired t test); # p <0.05 (paired t-test); ns, not significant; n is more than or equal to 5 mice/group, error + -SEM strip.

Figure 8 shows the in vivo effect of compound AHK2 on isoproterenol-induced cardiomyopathy in mdx mice.

The upper diagram: representative images of heart sections of control mice BL10(Ctl), mdx mice (mdx), and mdx mice treated with AHK2(mdx AHK 2). Calibration strip, 1 mm. Evans blue uptake can be seen by fluorescence microscopy.

The following figures: percentage of damaged areas in the heart of control mice (Ctl), mdx mice (mdx), and mdx mice treated with AHK2(mdx AHK 2). Analysis N-2 mice/group; error. + -. SEM strip.

Detailed Description

The present invention provides novel triazole compounds capable of treating or preventing a disorder or disease associated with dysregulation of intracellular calcium or dysfunction of RyR receptors.

In this sense, as mentioned above, the first aspect of the invention relates to compounds of formula (I):

wherein:

R ² is C ₁ -C ₆ Alkylene diradicals of which 1,2 or 3 are-CH ₂ The radicals may optionally be selected fromSubstitution of groups from-O-and-S-; and wherein C ₁ -C ₆ The alkylene diradicals may optionally be independently selected from C _1-4 Alkyl, allyl, propargyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 3-aminopropyl, 4-aminobutyl, 3-guanidinopropyl, 3-indolylmethyl, C _6-10 Aryl, benzyl, 4-hydroxybenzyl, C _6-10 Heteroaryl, F, Cl, OH, O (C) _1-4 Alkyl), CN, NO ₂ 、CO(C _1-4 Alkyl), CO ₂ (C _1-4 Alkyl), -CONH (C) _1-4 Alkyl), -CON (C) _1-4 Alkyl radical) ₂ Substituted with one or more groups of (a);

m is selected from 0,1, 2,3 and 4;

n is selected from 0,1 and 2;

y is selected from-OH and-CO ₂ H、-CO ₂ (C _1-4 Alkyl), -CO ₂ (allyl), -CO ₂ (benzyl), -SO ₃ H、-NH ₂ 、-NH(C _1-4 Alkyl), -N (C) _1-4 Alkyl radical) ₂ And N (C) _1-4 Alkyl radical) ₃ and-N (heterocyclyl or heteroaryl), wherein said heterocyclyl or heteroaryl is optionally substituted with C _1-4 Alkyl substituted, and wherein the N atom is part of a heterocyclyl or heteroaryl group;

or a pharmaceutically acceptable stereoisomer, salt, solvate or complex thereof, or an isotopically-labeled derivative thereof, or a prodrug thereof.

In the context of the present invention, the following terms appearing in the compounds of the formula (I) have the meanings indicated below:

the term "alkylenebisRadical "refers to such a double radical: formed by a linear or branched hydrocarbon chain consisting of carbon and hydrogen atoms, has no unsaturation and its ends are bound to the rest of the molecule by single bonds, such as, for example, methylene, ethylene, propylene, butylene, etc. C ₁ -C ₄ Reference to an alkylene diradical refers to the diradical having between 1 and 4 carbon atoms, and C ₁ -C ₆ Reference to an alkylene diradical refers to the diradical having between 1 and 6 carbon atoms. The alkylene diradicals may be substituted, as in the compounds of formula (I) R ¹ And R ² The definition of the substituents is as described.

The term "alkyl" refers to such a radical: formed by a linear or branched hydrocarbon chain consisting of carbon and hydrogen atoms, does not contain any saturation and is bound to the rest of the molecule by a single bond (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, etc.). C ₁ -C ₄ Reference to an alkyl group refers to the radical having between 1 and 4 carbon atoms.

The term "C ₆ -C ₁₀ Aryl "refers to a radical formed by a 6 to 10 membered aromatic ring consisting of carbon and hydrogen atoms, preferably a phenyl radical.

The term "C ₆ -C ₁₀ Heteroaryl "refers to a radical formed by a 6-10 membered aromatic ring consisting of carbon and hydrogen atoms and one or more heteroatoms selected from O, N and S.

The term "-N (heterocyclyl)" refers to a radical formed by a 5-to 7-membered ring consisting of carbon and hydrogen atoms and one or more heteroatoms selected from O, N and S, at least one of the heteroatoms being N and the latter being directly bound to R ² A free radical.

The term "-N (heteroaryl)" refers to a radical formed by a 6 to 10 membered aromatic ring consisting of carbon and hydrogen atoms and one or more heteroatoms selected from O, N and S, at least one of the heteroatoms being N and the latter being directly bound to R ² A free radical.

The term "allyl" refers toformula-CH ₂ -CH＝CH ₂ The radical of (1).

The term "halogen" refers to F, Cl, Br or I.

The expression "isotopically labelled derivative" refers to a compound of formula (I): wherein at least one of its atoms is enriched in an isotope. For example, wherein hydrogen is replaced by deuterium or tritium, carbon is enriched ¹³ C or ¹⁴ Replacement of C atoms, or enrichment of nitrogen ¹⁵ Compounds of formula (I) wherein the N atom is replaced are within the scope of the present invention.

The term "pharmaceutically acceptable salt or solvate" refers to any pharmaceutically acceptable salt, ester, solvate, or any other compound that, when administered to a recipient, is capable of providing (directly or indirectly) a compound of formula (I) as described herein. The salts may be prepared by methods known in the art.

For example, pharmaceutically acceptable salts of the compounds provided in this document are synthesized from compounds comprising the aforementioned basic or acidic units by conventional chemical methods. Such salts are generally prepared by: for example, the free acid or base forms of these compounds are reacted with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Nonaqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally preferred. Examples of acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate; and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate salts. Examples of base addition salts include inorganic salts such as, for example, sodium, potassium, calcium, ammonium, magnesium, aluminum, and lithium; and organic base salts such as, for example, ethylenediamine, ethanolamine, N-dialkyleneethanolamine, glucosamine, and basic amino acid salts.

Solvates refer to salts of the compounds of formula (I) in which the crystal lattice incorporates a molecule of a pharmaceutically suitable solvent. Solvation processes are well known in the art. Examples of pharmaceutically suitable solvents are ethanol, water and the like. In a specific embodiment, the solvate is a hydrate.

The compound of formula (I) or a salt or solvate thereof is preferably in a pharmaceutically acceptable form or in a substantially pure form. A pharmaceutically acceptable form is especially understood to have a pharmaceutically acceptable level of purity, excluding usual pharmaceutical additives such as diluents and excipients, and excluding any material that is considered toxic at normal dosage levels. The purity level of the drug is preferably above 50%, more preferably above 70%, even more preferably above 90%. In a preferred embodiment, the compound of formula (I) or a salt or solvate thereof has a purity level of 95% or more.

The compounds of the invention represented by formula (I) above may include any stereoisomer, including enantiomers and diastereomers, depending on the presence of a chiral center. Individual isomers, enantiomers or diastereomers and mixtures thereof are within the scope of the invention.

The term "prodrug" is used in its broadest sense and includes those derivatives that are converted in vivo to the compounds of the invention. Such derivatives include, without limitation, esters, amino acid esters, phosphate esters, metal salt sulfonates, carbamates, and amides depending on the functional groups present in the molecule. Examples of methods for preparing prodrugs of a given active compound are known to the person skilled in the art and can be found, for example, in Krogsgaard-Larsen et al, "Textbook of Drug Design and Discovery" Taylor & Francis (4 months 2002).

In one variant (A) of the invention, R ¹ The radical being-CH ₂ -。

In a preferred embodiment of said variant (A), R ² Is optionally substituted with a group independently selected from methyl, ethyl, propyl, isopropyl, allyl, propargyl, butyl, isobutyl, sec-butyl, tert-butyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 3-aminopropyl, 4-aminobutyl, 3-guanidinopropyl, 3-indolylmethyl, phenyl, 1-naphthyl, 2-naphthyl, benzyl, 4-hydroxybenzyl, and C _6-10 C substituted by one or two substituents of heteroaryl _1-4 Alkylene bis-freeAnd (4) a base.

More preferably, R ² is-CH ₂ -or-CH ₂ -CH ₂ -a diradical optionally substituted with one or two substituents independently selected from methyl, isopropyl, isobutyl and benzyl. Even more preferably, R ² is-CH optionally substituted with two methyl substituents or with one substituent selected from the group consisting of isopropyl, isobutyl and benzyl ₂ -a diradical, or R ² is-CH ₂ -CH ₂ -a double free radical. Also preferred, R ² is-CH ₂ -or-CH ₂ -CH ₂ -a double free radical.

In another preferred embodiment of said variant (A), R ³ Is H.

In another preferred embodiment of said variant (a), m is 1.

In another preferred embodiment of said variant (a), n is 0.

In another preferred embodiment of said variant (A), X is-O (C) _1-4 Alkyl) or halogen, more preferably meta or para methoxy, or chloro.

In another preferred embodiment of said variant (A), Y is chosen from CO ₂ H，CO ₂ Me，NH ₂ ，-NHMe，-NMe ₂ ，-NMe ₃ ，-NHEt，-NEt ₂ ，-NEt ₃ ，

Or a pharmaceutically acceptable salt of said group.

More preferably, Y is selected from CO ₂ H、CO ₂ Me、NH ₂ 、-NHMe、-NMe ₂ 、-NMe ₃ 、-NHEt、-NEt ₂ 、-NEt ₃ Pyrrolidin-1-yl, piperidin-1-yl, morpholin-4-yl, 4-piperazin-1-yl, 4-methyl-piperazin-1-yl, pyridin-1-yl, more preferably CO ₂ H and-NMe ₂ Even more preferably Y is-CO ₂ H or a pharmaceutically acceptable salt thereof.

In variant (a), the compound of formula (I) is selected from:

1-carboxymethyl-4- [3- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

1- [2- (N, N-dimethylamino) ethyl ] -4- [4- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

1-carboxymethyl-4- [4- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

(S) -1- (1-carboxy-2-phenylethyl) -4- [3- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

(R) -1- (1-carboxy-2-phenylethyl) -4- [3- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

(S) -1- (1-carboxy-3-methylbutyl) -4- [3- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

(R) -1- (1-carboxy-3-methylbutyl) -4- [3- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

(S) -1- (1-carboxy-2-methylpropyl) -4- [3- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

(R) -1- (1-carboxy-2-methylpropyl) -4- [3- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

1- (1-carboxy-1-methylethyl) -4- [3- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

1- (2-hydroxyethyl) -4- [4- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

1-methoxycarbonylmethyl-4- (phenylsulfinylmethyl) -1H-1,2, 3-triazole, and

1-carboxymethyl-4- [3- (methoxy) phenylsulfonylmethyl ] -1H-1,2, 3-triazole,

1-carboxymethyl-4- [3- (chloro) phenylthiomethyl ] -1H-1,2, 3-triazole,

Or a salt thereof.

In variant (B) of the invention, R ¹ The free radical being-CH ₂ -。

In a preferred embodiment of said variant (B), R ² Is C _1-4 Alkylene diradicals of which one or two is-CH ₂ -the group is replaced by-O-, and wherein said C ₁ -C ₄ Alkylene diradicals optionally substituted with one or two C ₁ -C ₄ Alkyl (preferably selected from methyl, ethyl, n-propyl, isopropyl, n-butyl and tert-butyl) substituted.

More preferably, R ² is-CH ₂ -or-CH ₂ -CH ₂ -a diradical optionally substituted with one or two substituents independently selected from methyl, isopropyl and isobutyl. Even more preferably, R ² is-CH optionally substituted with two methyl substituents or with one substituent selected from isopropyl and isobutyl ₂ -a diradical, or R ² is-CH ₂ -CH ₂ -a double free radical.

In another preferred embodiment of said variant (B), R ³ Is H.

In another preferred embodiment of said variant (B), m is 1.

In another preferred embodiment of said variant (B), n is 0.

In another preferred embodiment of said variant (B), X is-O (C) _1-4 Alkyl) or halogen, more preferably meta or para methoxy, or chloro.

In another preferred embodiment of said variant (B), Y is chosen from NH ₂ ，-NH(C ₁ -C ₄ Alkyl group), -N (C) ₁ -C ₄ Alkyl radical) ₂ ，-N(C ₁ -C ₄ Alkyl radical) ₃ ，

More preferably, Y is selected from-NH ₂ 、-NHMe、-NMe ₂ 、-NMe ₃ 、-NHEt、-NEt ₂ 、-NEt ₃ Pyrrolidin-1-yl, piperidin-1-yl, morpholin-4-yl, 4-piperazin-1-yl, 4-methyl-piperazin-1-yl, pyridin-1-yl, or a pharmaceutically acceptable salt of said group. More preferably, Y is-NMe ₂ Or a pharmaceutically acceptable salt thereof.

In variant (C) of the invention, R ¹ The free radical being-CH ₂ -。

In a preferred embodiment of said variant (C),R ² is optionally substituted with a group independently selected from methyl, ethyl, propyl, isopropyl, allyl, propargyl, butyl, isobutyl, sec-butyl, tert-butyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 3-aminopropyl, 4-aminobutyl, 3-guanidinopropyl, 3-indolylmethyl, phenyl, 1-naphthyl, 2-naphthyl, benzyl, 4-hydroxybenzyl, and C _6-10 C substituted by one or two substituents of heteroaryl _1-4 Alkylene diradicals.

More preferably, R ² is-CH ₂ -or-CH ₂ -CH ₂ -a diradical optionally substituted with one or two substituents independently selected from methyl, isopropyl, isobutyl and benzyl. Even more preferably, R ² is-CH optionally substituted with two methyl substituents or with one substituent selected from isopropyl, isobutyl and benzyl ₂ -a diradical, or R ² is-CH ₂ -CH ₂ -a double free radical. Also preferably, R ² is-CH ₂ -or-CH ₂ -CH ₂ -a double free radical.

In another preferred embodiment of said variant (C), R ³ Is H.

In another preferred embodiment of said variant (C), m is 1.

In another preferred embodiment of said variant (C), n is 0.

In another preferred embodiment of said variant (C), X is-O (C) _1-4 Alkyl) or halogen, more preferably meta or para methoxy, or chloro.

In another preferred embodiment of said variant (C), Y is chosen from CO ₂ H，CO ₂ (C ₁ -C ₄ Alkyl), NH ₂ ，-NH(C ₁ -C ₄ Alkyl group), -N (C) ₁ -C ₄ Alkyl radical) ₂ ，-N(C ₁ -C ₄ Alkyl radical) ₃ ，

Or a pharmaceutically acceptable salt of said group.

More preferably, Y is selected from-NH ₂ 、-NHMe、-NMe ₂ 、-NMe ₃ 、-NHEt、-NEt ₂ 、-NEt ₃ Pyrrolidin-1-yl, piperidin-1-yl, morpholin-4-yl, 4-piperazin-1-yl, 4-methyl-piperazin-1-yl, pyridin-1-yl, or a pharmaceutically acceptable salt of said group.

More preferably, Y is selected from CO ₂ H and-NMe ₂ Or a pharmaceutically acceptable salt thereof.

Method for obtaining the Compounds of the invention

The compounds of formula (I) of the present invention can be prepared by the following process: comprising reacting an alkyne of formula (II) with an azide of formula (III):

wherein R in the compounds of formulae (II) to (III) ¹ 、R ² 、R ³ M, n and X are as defined for the compound of formula (I), and wherein Y is a group as defined for the compound of formula (I), optionally protected with a carboxyl protecting group, a hydroxyl protecting group or an amino protecting group-depending on the nature of the Y group.

This reaction may be carried out in the presence of a copper catalyst such as, for example, copper (II) sulfate/sodium ascorbate, copper (I) iodide or copper (I) acetate.

Furthermore, in a preferred embodiment, the reaction between the compound of formula (II) and the compound of formula (III) is carried out in the presence of a base such as, for example, sodium acetate, diisopropylamine or triethylamine.

In a particular embodiment, when n is 0, the compound of formula (I) obtained according to the aforementioned process may be treated with an oxidizing agent to produce a compound of formula (I) wherein n is 1 or 2.

Examples of the oxidizing agent include, for example, 3-chloroperbenzoic acid or tert-butyl hydroperoxide.

In another embodiment, when the compound of formula (I) obtained according to the aforementioned process has protection with a protecting group(ii) when the group Y is present, removing the protecting group to yield a compound of formula (I): wherein R is ¹ 、R ² 、R ³ M, n, X and Y are as defined for the compounds of formula (I). The protecting groups can be removed according to methods known to those skilled in organic synthesis.

Pharmaceutical composition

The present invention further provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable stereoisomer, salt, solvate or complex thereof, or an isotopically labeled derivative thereof, or a prodrug thereof; and one or more pharmaceutically acceptable excipients or carriers.

A pharmaceutically acceptable carrier must be acceptable in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. The pharmaceutically acceptable carrier may be selected from the following organic and inorganic materials: are used in pharmaceutical formulations and are incorporated as analgesics, pH adjusting agents, linkers, disintegrants, diluents, emulsifiers, fillers, glidants, solubilizers, stabilizers, suspending agents, tonicity agents and thickeners. Pharmaceutical additives such as antioxidants, fragrances, dyes, flavor enhancers, preservatives and sweeteners may additionally be added.

Examples of the pharmaceutically acceptable carrier include carboxymethylcellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methylcellulose, saline solution, sodium alginate, sucrose, starch, talc, water and the like.

The pharmaceutical formulations of the present invention are prepared by methods well known in the pharmaceutical arts. For example, the compound of formula (I) is mixed with a pharmaceutically acceptable excipient or carrier as a suspension or solution. The choice of carrier is determined by the solubility and chemical nature of the compound, the chosen route of administration, and standard pharmaceutical practice.

The compounds or compositions of the present invention may be administered to a human or animal subject by any known method, including, without limitation, oral administration, sublingual or buccal administration, parenteral administration, transdermal absorption, by nasal drip or inhalation, vaginal, rectal and intramuscular administration.

In particular embodiments, parenteral administration is performed, such as, for example, by subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection. For parenteral administration, the compounds of the invention are combined with a sterile aqueous solution that is isotonic with the blood of the subject. Formulations of this type were prepared by: the solid active ingredient is dissolved in water containing a physiologically compatible substance such as sodium chloride, glycine and the like and having a buffered pH compatible with physiological conditions. The formulations are provided in single-dose or multi-dose containers, such as closed vials or ampoules.

In another embodiment, oral administration is performed. In this case, the formulation of the compound of the present invention may be provided in the form of capsules, tablets, powders, granules, or as a suspension or solution. The formulation may include conventional additives such as lactose, mannitol, starch, and the like; binders, such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch or gelatin; disintegrants, for example corn starch and potato starch or sodium carboxymethyl cellulose; lubricants, such as talc or magnesium stearate.

Applications of

The compounds of the invention are suitable for modulating RyR receptors that modulate calcium function in animal or human cells and are therefore capable of treating or preventing disorders or diseases associated with dysregulation of intracellular calcium substantially caused by dysfunction of RyR receptors.

By "dysregulation of intracellular calcium" is to be understood, in certain cases, dysregulation of calcium levels and calcium fluxes in the cell.

Thus, intracellular calcium (Ca) in quiescent conditions ²⁺ ) Increased concentrations lead to toxic muscle cell (muscle fiber) damage, with Ca ²⁺ Dependent proteases such as calpain are activated. If calpain activity is increased in necrotic muscle fibers in mdx mice and calpain dysfunction leads to limb girdle muscular dystrophy, by inhibiting intracellular Ca ²⁺ An increase to block the activity of calcium dependent proteases would result in the prevention of intramuscular atrophy and thus the treatment of diseases such as duchenne muscular dystrophy or becker muscular dystrophy.

In vitro assays using the compounds of the invention have demonstrated the ability of these compounds to increase the restoration of intracellular calcium in muscle fibers, suggesting that these compounds effect this restoration through mechanisms involved in the modulation of RyR channels. In addition, the compounds have also demonstrated the ability to reduce by half the number of overexpressed genes in dystrophic mdx muscle, one of which is associated with skeletal muscle loss or atrophy.

RyR receptors that regulate intracellular calcium function include RyR1, RyR2, and RyR3, as well as RyR proteins or RyR analogs. An RyR analog refers to a functional variant of a biologically active RyR protein having 60% or greater homology to the amino acid sequence of the RyR protein.

In the context of the present invention, "RyR bioactivity" is to be understood as being the protein or peptide activity which under the experimental conditions described herein shows the ability to physically associate (associate) or bind itself to FKBP12 (calpain-1) in the case of RyR1 and RyR3, and to physically associate or bind itself to FKBP12.6 (calpain-2) in the case of RyR 2.

The compounds of the invention are useful for limiting or preventing a decrease in RyR-binding FKBP (calponin) levels in a cell of a subject. Based on the foregoing, RyR bound FKBP refers to FKBP12 (calpain-1) bound to RyR1, FKBP12.6 (calpain-2) bound to RyR2, and FKBP12 (calpain-1) bound to RyR 3.

When the decrease is prevented, blocked, hindered, or reduced in any way by administration of a compound of the invention, the decrease in the level of RyR-bound FKBP in the cells of the subject is limited or prevented such that the level of RyR-bound FKBP in the cells of the subject is higher than in the absence of the administered compound.

The level of RyR binding FKBP in a subject is detected using: standard assays or techniques known to those skilled in the art, such as immunological techniques, hybridization assays, immunoprecipitation, western blot analysis, fluorescence imaging and/or radiation detection techniques, as well as any other assays or techniques, such as those disclosed in the experimental section of this document.

In particular embodiments, the decrease in level of RyR binding FKBP (calponin) occurs as a result of cells from the subject experiencing nitrooxidative stress. In fact, experimental tests carried out with the compounds of the invention have clearly shown that said compounds allow to minimize the degenerative effect of nitrooxidative stress on the myotubes of healthy humans through an increase in the affinity of the RyR 1-calpain 1 interaction.

Thus, it has been demonstrated that the compounds of the present invention prevent disorders or conditions involving the modulation of RyR receptors or the increase of intracellular calcium, which thereby allow the modulation of their levels. Examples of such disorders or conditions include skeletal muscle disorders and diseases (related to RyR1 modulation), cardiac disorders and diseases (related to RyR2 modulation), and nervous system disorders and diseases (related to RyR1, RyR2, or RyR3 modulation).

Thus, a further aspect of the present invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable stereoisomer, salt, solvate or complex thereof, or an isotopically labelled derivative thereof, or a prodrug thereof, for the manufacture of a pharmaceutical product for the treatment and/or prevention of skeletal muscle disorders and diseases, cardiac disorders and diseases and disorders and diseases of the nervous system.

In particular embodiments, the skeletal muscle disorders and diseases are selected from muscular dystrophy, congenital myopathy, metabolic myopathy, and intramuscular atrophy. Preferably, the skeletal muscle disorder or disease is duchenne muscular dystrophy or becker muscular dystrophy.

In another embodiment, the cardiac disorder and disease is selected from the group consisting of heart failure, cardiac ischemia, cardiac arrhythmia, and cardiomyopathy.

In another embodiment, the neurological disorders and diseases are selected from stroke, alzheimer's disease, frontotemporal dementia and cognitive impairment.

In a preferred embodiment, the compounds according to variant (a) of the invention are those used for the preparation of pharmaceutical products intended for the treatment of skeletal muscle disorders and diseases, as well as for the treatment of cardiac disorders and diseases, such as those mentioned above.

In another preferred embodiment, the compounds according to variant (B) of the invention are those used for the preparation of pharmaceutical products intended for the treatment of neurological disorders and diseases, such as those mentioned above.

Further aspects of the invention relate to compounds of formula (I), or a pharmaceutically acceptable stereoisomer, salt, solvate or complex thereof, or an isotopically labeled derivative thereof, or a prodrug thereof, for use in the treatment and/or prevention of skeletal muscle disorders and diseases, cardiac disorders and diseases, and nervous system disorders and diseases.

Another aspect of the present invention relates to a method for the treatment and/or prophylaxis of skeletal muscle disorders and diseases, cardiac disorders and diseases, and nervous system disorders and diseases, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable stereoisomer, salt, solvate or complex thereof, or an isotopically labeled derivative thereof, or a prodrug thereof.

A "therapeutically effective" amount is certainly understood to be an amount sufficient to obtain a beneficial or desired result (prophylactic and/or therapeutic response, prevention or substantial alleviation of undesired side effects).

In particular embodiments, the compounds of the present invention are administered to a subject in an amount effective to modulate aberrant intracellular calcium concentrations. This amount is readily determined by one skilled in the art using known methods. For example, release of intracellular calcium through the RyR channel can be quantified using a calcium-sensitive fluorescent dye such as Fluo-3 or Fura-2 and monitoring the calcium-related fluorescent signal with a photomultiplier tube and appropriate software (Brillants, et al. cell,1994,77,513- "Stabilization of calcium (ryanodine receptor) function by FK506-binding protein"; Gillo, et al. blood,1993,81, 783-.

The concentration of a compound of the invention in the serum of a human or animal subject may be determined according to methods known in the art (theris, m.et al. drug test. analysis 2009,1,32-42 "Screening for the calstabin-ryanodine receptors modulators JTV-519and S-107in therapy control analysis"). The amount of a compound of formula (I) administered that is effective to limit or prevent abnormal intracellular calcium levels will depend on the relative potency of the selected compound, the severity of the disorder being treated and the body weight of the affected subject. However, the compounds will generally be administered once or more a day, for example 1,2,3 or 4 times daily, with a total typical daily dose in the following ranges: between about 1 mg/kg/day and 100 mg/kg/day, more preferably between 10 mg/kg/day and 40 mg/kg/day, or an amount sufficient to obtain a serum level of between about 1ng/ml and 500 ng/ml.

The compounds of formula (I) may be used alone, in combination with each other, or in combination with other agents having therapeutic activity including, but not limited to, mRNA exon splicing enhancers, gene transcription modulators, diuretics, anticoagulants, platelet agents, antiarrhythmics, inotropic agents, chronotropic agents, alpha-and beta-blockers, angiotensin inhibitors, and vasodilators.

The medicaments may be part of the same composition, or may be provided as separate compositions-for administration at the same time or at different times.

The invention also includes an in vitro method for determining the ability of a compound to modulate intracellular calcium levels and prevent dissociation of calponin from the RyR protein complex, wherein the method comprises: (a) obtaining or producing a cell culture comprising an RyR receptor; (b) contacting the cell with a compound to be tested; (c) exposing the cell to one or more known conditions that alter intracellular calcium regulation or produce post-translational modifications in the RyR receptor; (d) determining whether the compound modulates intracellular calcium levels; and (e) determining whether the compound limits or prevents dissociation of calpain from the RyR protein complex.

In particular embodiments, the condition that alters intracellular calcium regulation or produces a post-translational modification in the RyR receptor is oxidative stress or nitrosative stress.

The present invention also contemplates a method for diagnosing a disorder or disease, wherein the method comprises:

-obtaining a tissue or cell sample comprising RyR receptors from a subject;

incubating the tissue or cell sample obtained in step a) with a compound of formula (I), and

-determining:

(a) whether the RyR-calpain interaction is increased relative to RyR-calpain interaction in a control cell or tissue;

or

(b) Whether intracellular calcium levels are reduced, as compared to the absence of such a reduction in control cells or tissues;

wherein an increase in the RyR-calpain interaction in (a) or a decrease in the intracellular calcium level in (b) is indicative of the presence of the disorder or disease in the subject.

In a particular embodiment, the compound of formula (I) used in the diagnostic method is a compound according to variant (C) of the invention.

In another embodiment, the tissue sample is a muscle tissue sample.

In a specific embodiment, when RyR is RyR1, the disorder or disease to be diagnosed is a skeletal muscle disorder or disease.

In a specific embodiment, when RyR is RyR2, the disorder or disease to be diagnosed is a cardiac disorder or disease.

In a specific embodiment, when RyR is RyR1, RyR2, or RyR3, the disorder or disease to be diagnosed is a neurological disorder or disease.

The increase in RyR-calpain interactions and decrease in intracellular calcium levels can be measured by techniques known to the skilled artisan, such as immunoprecipitation, proximity ligation assays in situ (PLA), and real-time calcium imaging using fluorescent probes.

Examples

The acronyms for the compounds, reagents, solvents or techniques used are defined as follows:

-AHK 1: a compound according to formula (I) comprising the group: r ¹ ＝R ² ＝–CH ₂ –；R ³ ＝H；m＝1；n＝0；X＝3-MeO；Y＝CO ₂ H，

-AHK 2: a compound according to formula (I) comprising the group: r ¹ ＝–CH ₂ –；R ² ＝–CH ₂ CH ₂ –；R ³ ＝H；m＝1；n＝0；X＝4-MeO；Y＝NMe ₂ ，

-S-107: the structures of compounds used for comparative purposes in biological tests that have been performed are described in the background section of this document.

- ^t BuOH: the concentration of the tertiary butanol is controlled by the concentration of the tertiary butanol,

-DAPI: 4', 6-diamino-2-phenylindole,

-ESI: the ionization is carried out by the electric injection,

-EtOAc: the reaction solution is mixed with ethyl acetate to prepare ethyl acetate,

-HRMS: (ii) high-resolution mass spectrometry,

-IR: an infrared spectroscopic method is adopted to carry out the infrared spectroscopic method,

-mdx: an animal model with X duchenne muscular dystrophy,

-MP: the melting point of the compound is shown in the specification,

-NMR: the Nuclear Magnetic Resonance (NMR) of the sample,

-SIN 1: 3-morpholinyl-sydnimine (sydnonimine), peroxynitrous acid (yl), and a nitric oxide generating agent.

-THF: the reaction mixture of tetrahydrofuran and tetrahydrofuran is prepared by reacting tetrahydrofuran,

-TBTA: tris [ (1-benzyl-1H-1, 2, 3-triazol-4-yl) methyl ] amine.

The following examples are provided for illustrative purposes and are not intended to limit the invention.

Example 1: 1-methoxycarbonylmethyl-4- [4- (methoxy) phenylthiomethyl]-1H-1,2, 3-triazole

Preparation of 1- (4-Methoxyphenylthio) -2-propyne (2mmol, 356mg), methyl azidoacetate (2.00mmol, 230mg) and TBTA (catalyst) in THF under a Nitrogen atmosphere ^t BuOH (1: 1, 4mL) solution. Two degassed aqueous solutions were then added: CuSO ₄ (0.4mmol, 63mg) in H ₂ O (1mL), and sodium ascorbate (0.8mmol, 158mg) in H ₂ O (1 mL); and the mixture was stirred at room temperature overnight. After completion of the reaction, the solvent was evaporated, 28% aqueous ammonia (10mL) was added, and CH was used ₂ Cl ₂ The product was extracted (3X 20 mL). Drying (MgSO) ₄ ) The organic extracts were combined and the solvent was evaporated under reduced pressure. The product was purified by column chromatography (silica gel; 1: 3 EtOAc/hexane). Yield: 477mg (78%). A light yellow oil. IR (cm- ¹ ):2953,2837(C-H) 1750(C ═ O),1219,1174 (triazole). ¹ H NMR(500MHz，CDCl ₃ ):δ7.38(s,1H),7.30(d,J＝8.7Hz,2H),6.81(d,J＝8.7Hz,2H),5.09(s,3H),4.12(s,2H),3.78(s,3H),3.77(s,3H)。 ¹³ C NMR(125MHz，CDCl ₃ ):δ166.7,159.4,145.8,134.0,125.4,123.5,114.7,55.4,53.1,50.8,30.9。C ₁₃ H ₁₆ N ₃ O ₃ HRMS (ESI +, m/z) for S, calculated: 294.0912, respectively; detection value: 294.0916.

example 2: 1-methoxycarbonylmethyl-4- [3- (methoxy) phenylthiomethyl]-1H-1,2, 3-triazole

Starting from 1- (3-methoxyphenylthio) -2-propyne (2.00mmol, 356mg) and methyl azidoacetate (2.00mmol, 230mg), the procedure is as for example 1. Yield: 537mg (88%). A light yellow oil. IR (cm) ^–1 ):2953,2837(C-H),1749(C ═ O),1220,1180 (triazole). ¹ H NMR(500MHz,CDCl ₃ ):δ7.51(s,1H),7.18(t,J＝8.0Hz,1H),6.92(d,J＝7.7Hz,1H),6.90-6.87(m,1H),6.73(dd,J＝8.2,1.8Hz,1H)5.11(s,2H),4.26(s,2H),3.78(s,3H),3.77(s,3H)。 ¹³ C NMR(125MHz,CDCl ₃ ):δ166.7,159.9,145.7,136.8,129.9,123.6,121.5,114.6,112.5,55.4,53.1,50.8,28.7。C ₁₃ H ₁₆ N ₃ O ₃ HRMS (ESI +, m/z) for S, calculated: 294.0912; detection value: 294.0917.

example 3: (S) -1- (1-methoxycarbonyl-2-phenylethyl) -4- [3- (methoxy) phenylthiomethyl]-1H- 1,2, 3-triazoles

Starting from 1- (3-methoxyphenylthio) -2-propyne (2.00mmol, 384mg) and methyl (S) -2-azido-3-phenylpropionate (2.00mmol, 358mg), the procedure is as for example 1. Yield of the product: 668mg (87%). A light yellow oil. [ alpha ] to] _D ²⁵ ＝-56.1°(c.1.01g/100mL,CH ₂ Cl ₂ )。IR(cm- ¹ ) 2952,2836(C-H),1744(C ═ O),1228,1172 (triazole). ¹ H NMR(400MHz,CDCl ₃ ):δ7.47(s,1H),7.27-6.62(m,9H),5.50(dd,J＝8.5,6.2Hz,1H),4.12(q,J＝14.9Hz,2H),3.75(s,3H),3.73(s,3H),3.46(dd,J＝14.0,5.8Hz,1H),3.36(dd,J＝13.9,9.2Hz,1H)。 ¹³ C NMR(101MHz,CDCl3):δ168.6,159.9,145.1,136.8,134.7,129.9,128.9,127.6,122.4,121.5,114.5,112.5,64.3,55.4,53.2,38.9,28.7。C ₂₀ H ₂₂ N ₃ O ₃ HRMS (ESI +, m/z) for S, calculated: 384.1382, respectively; detection value: 384.1392.

example 4: (R) -1- (1-methoxycarbonyl-2-phenylethyl) -4- [3- (methoxy) phenylthiomethyl]-1H- 1,2, 3-triazoles

Starting from 1- (3-methoxyphenylthio) -2-propyne (2.00mmol, 384mg) and (R) -methyl 2-azido-3-phenylpropionate (2.00mmol, 358mg), the procedure is as for example 1. Yield: 653mg (85%). A light yellow oil. [ alpha ] to] _D ²⁵ ＝+39.4°(c.2.71g/100mL,CH ₂ Cl ₂ )。C ₂₀ H ₂₂ N ₃ O ₃ HRMS (ESI +, m/z) for S, calculated: 384.1382; detection value: 384.1382. the NMR data were the same as in example 3.

Example 5: (S) -1- (1-methoxycarbonyl-3-methyl-butyl) -4- [3- (methoxy) phenylthiomethyl]-1H- 1,2, 3-triazoles

Starting from 1- (3-methoxyphenylthio) -2-propyne (2.00mmol, 384mg) and (S) -2-azido-4-methylpentanoic acid methyl ester (2.00mmol, 342mg)The procedure of example 1 was followed. Yield: 677.9mg (97%). A light yellow oil. [ alpha ] to] _D ²⁵ ＝+10.7°(c.1.00g/100mL,CH ₂ Cl ₂ )。 ¹ H NMR(500MHz,CDCl ₃ ):δ7.51(s,1H),7.16(t,J＝8.0Hz,1H),6.90(d,J＝7.7Hz,1H),6.87(s,1H),6.72(dd,J＝8.2,1.8Hz,1H),5.38(t,J＝8.0Hz,1H),4.24(q,J＝14.8Hz,2H),3.76(s,3H),3.73(s,3H),1.94(t,J＝7.5Hz,2H),1.19(dt,J＝13.4,6.7Hz,1H),0.90(d,J＝6.5Hz,3H),0.84(d,J＝6.6Hz,3H)。 ¹³ C NMR(125MHz,CDCl ₃ ):δ169.8,159.9,145.3,136.6,129.8,122.1,115.1,112.7,61.1,55.3,53.1,41.4,29.1,24.7,22.7,21.3。

Example 6: (R) -1- (1-methoxycarbonyl-3-methyl-butyl) -4- [3- (methoxy) phenylthiomethyl]-1H- 1,2, 3-triazoles

Starting from 1- (3-methoxyphenylthio) -2-propyne (2.00mmol, 384mg) and (R) -2-azido-4-methylpentanoic acid methyl ester (2.00mmol, 342mg), the procedure is as in example 1. Yield: 653mg (93%). A pale yellow oil. [ alpha ] to] _D ²⁵ ＝-12.1°(c.1.03g/100mL,CH ₂ Cl ₂ ). The NMR data were the same as in example 5.

Example 7: (S) -1- (1-methoxycarbonyl-2-methyl-propyl) -4- [3- (methoxy) phenylthiomethyl]-1H- 1,2, 3-triazoles

Starting from 1- (3-methoxyphenylthio) -2-propyne (2.00mmol, 384mg) and (S) -2-azido-3-methylbutanoic acid methyl ester (2.00mmol, 314mg), the procedure is as in example 1. Yield: 616mg (92%). A light yellow oil. [ alpha ] of] _D ²⁵ ＝+21.5°(c.1.03g/100mL,CH ₂ Cl ₂ )。 ¹ H NMR(500MHz,CDCl ₃ ):δ7.63(s,1H),7.16(t,J＝7.9Hz,1H),6.92(d,J＝7.5Hz,1H),6.88(s,1H),6.72(d,J＝6.8,1H),5.06(d,J＝8.7Hz,1H),4.24(q,J＝14.7Hz,2H),3.76(s,6H),2.44-2.30(m,1H),0.96(d,J＝6.6Hz,3H),0.73(d,J＝6.6Hz,3H)。 ¹³ C NMR(125MHz,CDCl ₃ ):δ168.8,159.6,144.7,136.3,129.5,121.9,115.0,112.3,68.5,55.0,52.5,32.0,28.8,18.8,18.1。

Example 8: (R) -1- (1-methoxycarbonyl-2-methyl-propyl) -4- [3- (methoxy) phenylthiomethyl]-1H- 1,2, 3-triazoles

Starting from 1- (3-methoxyphenylthio) -2-propyne (2.00mmol, 384mg) and (R) -2-azido-3-methylbutanoic acid methyl ester (2.00mmol, 314mg), the procedure is as in example 1. Yield: 626mg (93%). A light yellow oil. [ alpha ] to] _D ²⁵ ＝-19.5°(c.1.06g/100mL,CH ₂ Cl ₂ ). The NMR data were the same as in example 7.

Example 9: 4- [3- (methoxy) phenylthiomethyl group]-1- (1-methyl-1-methoxycarbonylethyl) -1H-1,2, 3-triazoles

Starting from 1- (3-methoxyphenylthio) -2-propyne (2.00mmol, 384mg) and methyl 2-azidoisobutyrate (2.00mmol, 386mg), the procedure is as for example 1. Yield: 258mg (40%). A light yellow oil. ¹ H NMR(400MHz,CDCl ₃ ):δ7.44(s,1H),7.11(t,J＝7.7Hz,1H),6.85(d,J＝7.6Hz,1H),6.82(s,1H),6.67(d,J＝7.6Hz,1H),4.17(s,2H),3.69(s,3H),3.62(s,3H),1.82(s,6H)。 ¹³ C NMR(101MHz,CDCl ₃ )δ171.6,159.7,144.3,136.7,129.6,121.6,121.0,114.8,112.3,64.3,55.1,53.1,28.8,25.5。

Example 10: 1-(2-hydroxyethyl) -4- [4- (methoxy) phenylthiomethyl]-1H-1,2, 3-triazole

Starting from 1- (4-methoxyphenylthio) -2-propyne (2.00mmol, 384mg) and 2-azidoethanol (2.00mmol, 174mg), the procedure is as in example 1. Yield: 520mg (98%). A light yellow oil. ¹ H NMR(400MHz,CDCl ₃ )δ7.40(s,1H),7.29(d,J＝8.3Hz,2H),6.80(d,J＝8.2Hz,2H),4.40(s,2H),4.09(s,2H),3.99(s,2H),3.77(s,3H),3.18(s,1H)。 ¹³ C NMR(101MHz,CDCl ₃ ):δ159.5,144.9,133.9,125.4,123.5,114.8,61.1,55.5,52.9,30.9。C ₁₂ H ₁₅ N ₃ O ₂ HRMS (ESI +, m/z) for S, calculated: 266.0963, respectively; detection value: 266.0965.

example 11: 1-carboxymethyl-4- [4- (methoxy) phenylthiomethyl]-1H-1,2, 3-triazole

Lithium hydroxide monohydrate (2.00mmol, 84mg) was added to 1-methoxycarbonylmethyl-4- [4- (methoxy) phenylthiomethyl]THF/H of (E) -1H-1,2, 3-triazole (1.00mmol, 305mg, example 1) ₂ O (1: 1, 8mL), and the resulting mixture was stirred at room temperature for 1 hour. The organic solvent was evaporated, the resulting water mixture was acidified with 1M HCl, and the solution was extracted with EtOAc (2 × 10 mL). Drying (MgSO) ₄ ) The organic phases were combined and the solvent was evaporated under reduced pressure. Yield: 158mg (54%). A white solid. 163 ℃ and 170 ℃. IR (cm) ^-1 ) 2923,2848(C-H),1730(C ═ O),1223,1188 (triazole). ¹ H NMR(500MHz,CD ₃ OD):7.66(s,1H),7.28(d,J＝8.8Hz,2H),6.84(d,J＝8.8Hz,2H),5.19(s,2H),4.07(s,2H),3.76(s,3H)。 ¹³ C NMR(125MHz,CD ₃ OD):δ169.7,161.1,146.3,135.6,126.3,115.7,55.8,51.6,31.5。C ₁₂ H ₁₄ N ₃ O ₃ HRMS (ESI +, m/z) for S, calculated: 280.0756; detection value: 280.0760.

example 12: 1-carboxymethyl-4- [3- (methoxy) phenylthiomethyl]-1H-1,2, 3-triazole

From 1-methoxycarbonylmethyl-4- [3- (methoxy) phenylthiomethyl]Starting with-1H-1, 2, 3-triazole (1.00mmol, 305mg, example 2), the procedure is as in example 11. Yield: 274mg (94%). A white solid. MP 115-119 ℃. IR (cm) ^-1 ) 2999,2971(C-H),1707(C ═ O),1229 (triazole). ¹ H NMR(500MHz,CD ₃ OD):δ7.80(s,1H),7.18(t,J＝8.0Hz,1H),6.96-6.86(m,2H),6.76(dd,J＝8.3,1.8Hz,1H),5.19(s,2H),4.23(s,2H),3.75(s,3H)。 ¹³ C NMR(125MHz,CD ₃ OD):δ169.7,161.4,146.1,138.0,130.9,125.9,123.1,116.1,113.6,55.7,51.7,29.3。C ₁₂ H ₁₄ N ₃ O ₃ HRMS (ESI +, m/z) for S, calculated: 280.0756, respectively; detection value: 280.0753.

example 13: (S) -1- (1-carboxy-2-phenylethyl) -4- [3- (methoxy) phenylthiomethyl]-1H-1,2,3- Triazole compounds

From (S) -1- (1-methoxycarbonyl-2-phenylethyl) -4- [3- (methoxy) phenylthiomethyl]Starting with-1H-1, 2, 3-triazole (1.00mmol, 383mg, example 3), the procedure of example 11 was followed. Yield: 318mg (86%). A white solid. MP is 79-83 ℃. [ alpha ] to] _D ²⁴ ＝-23.3°(c.1.12g/100mL,CH ₂ Cl ₂ )。IR(cm- ¹ ) 2931(C-H),1727(C ═ O),1246,1229 (triazole). ¹ H NMR(500MHz,CD ₃ OD):δ7.77(s,1H),7.20-6.71(m,9H),5.56(dd,J＝10.8,4.6Hz,1H),4.15(s,2H),3.73(s,3H),3.56(dd,J＝14.3,4.5Hz,1H),3.40(dd,J＝14.3,10.9Hz,1H)。 ¹³ C NMR(125MHz,CD ₃ OD):δ171.1,161,4,145.9,137.9,137.2,130.8,129.9,128.1,124.8,122.9,115.9,113.5,65.8,55.7,38.9,29.0。C ₂₀ H ₂₂ N ₃ O ₃ HRMS (ESI +, m/z) for S, calculated: 384.1382, respectively; detection value: 384.1392.

example 14: (R) -1- (1-carboxy-2-phenylethyl) -4- [3- (methoxy) phenylthiomethyl]-1H-1,2,3- Triazole compounds

From (R) -1- (1-methoxycarbonyl-2-phenylethyl) -4- [3- (methoxy) phenylthiomethyl]Starting with-1H-1, 2, 3-triazole (1.00mmol, 383mg, example 4), the procedure of example 11 was followed. Yield: 280mg (76%). A white solid. MP at 78-86 deg.c. C ₂₀ H ₂₂ N ₃ O ₃ HRMS (ESI +, m/z) for S, calculated: 384.1382; detection value: 384.1382. [ alpha ] of] _D ²⁴ ＝+16.5°(c.0.98g/100mL,CH ₂ Cl ₂ ). The NMR data were the same as in example 13.

Example 15: (S) -1- (1-carboxy-3-methylbutyl) -4- [3- (methoxy) phenylthiomethyl]-1H-1,2,3- Triazole compounds

From (S) -1- (1-methoxycarbonyl-3-methyl-butyl) -4- [3- (methoxy) phenylthiomethyl]Starting with-1H-1, 2, 3-triazole (1.00mmol, 349mg, example 5), the procedure is as in example 11. Yield: 265mg (76%). A white solid. MP at 96-105 ℃. [ alpha ] to] _D ^25.6 ＝+9.3°(c.1.11g/100mL,CH ₂ Cl ₂ )。 ¹ H NMR(500MHz,CDCl ₃ ):δ11.88(s,1H),7.54(s,1H),7.12(t,J＝7.9Hz,1H),6.92-6.77(m,2H),6.70(dd,J＝8.1,1.7Hz,1H),5.38(dd,J＝10.7,5.0Hz,1H),4.23(dd,J＝40.8,14.9Hz,2H),3.70(s,3H),2.03-1.86(m,2H),1.17(dt,J＝19.8,6.5Hz,1H),0.88(d,J＝6.5Hz,3H),0.82(d,J＝6.5Hz,3H)。 ¹³ CNMR(125MHz,CDCl ₃ ):δ171.2,159.8,144.7,135.9,129.8,122.6,122.4,115.6,112.9,61.8,55.3,41,2,28.5,24.7,22.6,21.1。

Example 16: (R) -1- (1-carboxy-3-methylbutyl) -4- [3- (methoxy) phenylthiomethyl]-1H-1,2,3- Triazole compounds

From (R) -1- (1-methoxycarbonyl-3-methyl-butyl) -4- [3- (methoxy) phenylthiomethyl]Starting with-1H-1, 2, 3-triazole (1.00mmol, 349mg, example 6), the procedure is as in example 11. Yield: 301mg (86%). A white solid. MP at 97-104 ℃. [ alpha ] to] _D ^25.4 ＝-12.1°(c.0.95g/100mL,CH ₂ Cl ₂ ). The NMR data were the same as in example 15.

Example 17: (S) -1- (1-carboxy-2-methylpropyl) -4- [3- (methoxy) phenylthiomethyl]-1H-1,2,3- Triazole

From (S) -1- (1-methoxycarbonyl-2-methyl-propyl) -4- [3- (methoxy) phenylthiomethyl]Starting with-1H-1, 2, 3-triazole (1.00mmol, 335mg, example 7) the procedure is as in example 11. Yield: 279mg (87%). A white solid. MP 115-121 ℃. [ alpha ] to] _D ^25.6 ＝+4.5°(c.1.06g/100mL,CH ₂ Cl ₂ )。 ¹ H NMR(500MHz,CDCl ₃ ):δ11.53(s,1H),7.68(s,1H),7.12(t,J＝7.8Hz,1H),6.87(d,J＝7.4Hz,1H),6.84(s,1H),6.70(d,J＝6.8Hz,1H),5.13(d,J＝7.6Hz,1H),4.23(dd,J＝43.5,14.5Hz,2H),3.71(s,3H),2.44(d,J＝6.4Hz,1H),0.96(d,J＝6.4Hz,3H),0.75(d,J＝6.4Hz,3H)。 ¹³ C NMR(125MHz,CDCl ₃ ):δ170.7,159.9,144.6,136.1,129.9,122.9,122.8,115.8,113.0,69.3,55.4,32.2,28.7,19.2,18.3。

Example 18: (R) -1- (1-carboxy-2-methylpropyl) -4- [3- (methoxy) phenylthiomethyl]-1H-1,2,3- Triazole

From (R) -1- (1-methoxycarbonyl-2-methyl-propyl) -4- [3- (methoxy) phenylthiomethyl]Starting with-1H-1, 2, 3-triazole (1.00mmol, 335mg, example 8), the procedure is as in example 11. Yield: 290mg (90%). A white solid. MP 114-. [ alpha ] to] _D ^25.6 ＝-6.7°(c.1.01g/100mL,CH ₂ Cl ₂ ). The NMR data were the same as in example 17.

Example 19: 1- (1-carboxy-1-methylethyl) -4- [3- (methoxy) phenylthiomethyl]-1H-1,2, 3-triazole

From 4- [3- (methoxy) phenylthiomethyl]Starting from (E) -1- (1-methyl-1-methoxycarbonylethyl) -1H-1,2, 3-triazole (0.8mmol, 258mg, example 9) the procedure of example 11 was followed. Yield: 246mg (100%). A pale yellow solid. MP:109-121 ℃. ¹ H NMR(400MHz,CD ₃ OD):δ7.77(s,1H),7.09(t,J＝8.0Hz,1H),6.82(d,J＝8.0Hz,1H),6.79(s,1H),6.67(d,J＝8.3Hz,1H),4.12(s,2H),3.65(s,3H),1.78(s,6H)。 ¹³ C NMR(101MHz,CD ₃ OD)δ174.2,161.3,145.3,137.9,130.8,123.4,116.5,113.7,65.9,55.7,29.5,25.9。

Example 20: 1- [2- (N, N-dimethylamino) ethyl]-4- [4- (methoxy) phenylthiomethyl group]-1H-1,2, 3-triazoles

Triethylamine (8.80mmol, 1.22mL) and methanesulfonyl chloride (4.39mmol, 0.34mL) were added successively under a nitrogen atmosphere to 1- (2-hydroxyethyl) -4- [4- (methoxy) phenylthiomethyl cooled at 0 deg.C]-1H-1,2, 3-triazole (2.92mmol, 776mg, example 10) in anhydrous THF (16 mL). The reaction mixture was stirred at room temperature overnight. Dimethylamine hydrochloride (8.00mmol, 652mg), triethylamine (8.80mmol, 1.22mL), NaI (0.13mmol, 20mg) and an additional amount of anhydrous THF (8mL) were then added and the mixture was stirred at 50 deg.C overnight. After completion of the reaction, the solvent was evaporated under reduced pressure, the resulting residue was dissolved in EtOAc (20mL) and saturated NaHCO was used ₃ The aqueous solution (30mL) and brine (10mL) were washed successively. Drying (MgSO) ₄ ) The organic layer was evaporated to dryness in vacuo. Yield: 572mg (84%). A white solid. MP at 35-40 deg.c. IR (cm- ¹ ) 2943(N-H),2860,2771(C-H),1242 (triazole). ¹ HNMR(400MHz,CDCl ₃ ):δ7.41(s,1H),7.30(d,J＝7.6Hz,2H),6.81(d,J＝7.8Hz,2H),4.37(s,2H),4.11(s,2H),3.77(s,3H),2.71(s,2H),2.25(s,6H)。 ¹³ C NMR(101MHz,CDCl ₃ )：159.2,144.8,133.7,125.5,122.6,114.5,58.6,55.3,48.0,45.3,30.9。C ₁₄ H ₂₁ N ₄ HRMS (ESI +, m/z) for OS, calculated: 293.1436, respectively; detection value: 293.1440.

example 21: 1-methoxycarbonylmethyl-4- (phenylsulfinylmethyl) -1H-1,2, 3-triazole

Starting from thiophenyl-2-propyne (2.00mmol, 268mg) and methyl azidoacetate (2.00mmol, 230mg), the procedure of example 1 was followed. Yield: 342mg (65%). A white solid. MP at 70-74 deg.c. IR (cm) ^–1 ) 2953,2837(C-H),1749(C ═ O),1220,1180 (triazole). ¹ H NMR(400MHz,CDCl ₃ ):δ7.49(s,1H),7.34(d,J＝7.6Hz,2H),7.31–7.22(m,2H),7.20(m,1H),5.11(s,2H),4.27(s,2H),3.78(s,3H)。 ¹³ CNMR(101MHz,CDCl ₃ ):δ166.5,144.6,135.1,129.1,128.6,126.1,123.5,52.6,50.3,28.3. M-chloroperbenzoic acid (1.00mmol, 172mg) was added to the resulting solution of 1- (methoxycarbonylmethyl) -4- (phenylthiomethyl) -1H-1,2, 3-triazole (1.00mmol, 263mg) in anhydrous chloroform (30mL) at 0 ℃ and the mixture was stirred at the same temperature for 30 minutes. The mixture was evaporated and the residue was purified by column chromatography (silica gel; 5: 95 MeOH/CH) ₂ Cl ₂ ). A colorless oil. Yield: 161mg (58%). ¹ H NMR(500MHz,CDCl ₃ ):δ7.72(s,1H),7.49(s,5H),5.24-5.10(dd,J＝5.13Hz,2H),4.29-4.15(dd,J＝4.28Hz,2H),3.82(s,3H)。

Example 22: 1-carboxymethyl-4- [3- (methoxy) phenylsulfonylmethyl]-1H-1,2, 3-triazole

M-chloroperbenzoic acid (2.50mmol, 437mg) was added to 1- (carboxymethyl) -4- [3- (methoxy) phenylthiomethyl at 0 deg.C]-1H-1,2, 3-triazole (1.00mmol, 279mg, example 12) in chloroform/acetonitrile 1: 1 anhydrous mixture (3mL) and the mixture was stirred at room temperature overnight. The mixture was evaporated and the residue was purified by column chromatography (silica gel; 5: 95 MeOH/CH) ₂ Cl ₂ ). Yield: 203mg (65%). A white solid. 106 ℃ and 115 ℃. ¹ H NMR(500MHz,CDCl ₃ ):7.91(s,1H),7.50-7.29(Ar,4H),4.99(s,2H),4.71(s,2H),3.84(s,3H)。C ₁₂ H ₁₄ N ₃ O ₅ HRMS (ESI +, m/z) for S, calculated: 312.0654; detection value: 312.0662.

example 23: 1-carboxymethyl-4- [3- (chloro) phenylthiomethyl]-1H-1,2, 3-triazole

A solution of bromoacetic acid (10mmol, 1.38g) and sodium azide (40mmol, 2.60g) in water (4mL) was stirred at room temperature overnight. Then neutralizing and dissolving with 3M NaOHLiquid, and acetonitrile (15mL), 1- (3-chlorophenylthio) -2-propyne (8mmol, 1.45g), sodium acetate (30mmol, 2.46g) and copper (I) acetate (2mmol, 242mg) were added successively. The resulting mixture was stirred at 45 ℃ for 6 h, the solvent was evaporated, acidified with 1M HCl and extracted with EtOAc (3X 20 mL). Drying (MgSO) ₄ ) The organic phases were combined and the solvent was evaporated under reduced pressure. Yield: 1.49g (66%). A white solid. MP 146 and 148 ℃. IR (cm- ¹ ) 2928,2850(C-H),1731(C ═ O),1224,1184 (triazole). ¹ H NMR(500MHz,CD ₃ OD):7.86(s,1H),7.39(s,1H),7.28-7.21(m,3H),5.22(s,2H),4.29(s,2H)。 ¹³ CNMR(125MHz,CD ₃ OD):δ168.5,144.1,137.9,134.4,130.0,128.8,127.6,126.3,124.6,50.4,27.7。C ₁₁ H ₁₀ ClN ₃ O ₂ HRMS (ESI +, m/z) for S, calculated: 283.0182, respectively; detection value: 283.0190.

example 24: in vitro toxicity biological assay in human cells

These experiments were performed in human control myotubes LHCN-M2 after 14 days in differentiation medium. To determine the toxicity of the different compounds analyzed (specifically, AHK1 and AHK2 according to the present invention), these compounds were added to the medium at different concentrations (0-2mM) for 24 hours at 37 ℃. For comparison purposes, the same experiment was performed by adding the reference compound S-107. Cell viability was determined by Cytotox 96(Promega) colorimetric assay according to the instructions in the manual.

The results are shown in figure 1, where it can be seen that the compounds according to the invention do not show acute toxicity in vitro on human myotubes at concentrations between 10nM and 2 mM. The performance of the compounds AHK1 and AHK2 after 24 hours of incubation was compared to the 100% cytotoxicity of the reference compound S-107 at a concentration of 1mM under the same conditions. This result demonstrates that the compounds according to the invention are less toxic than S-107, indicating that AHK compounds may be a better choice for therapeutic treatment of disorders involving abnormal calcium homeostasis in humans.

Example 25 in vitro assay for determining intracellular calcium levels in mouse muscle fibers

Will make the mouse toe flexor brevisIsolated fibers were cultured overnight in the presence or absence of compounds AHK1 and AHK2 at a concentration of 150 nM. Baseline intracellular calcium levels were assessed by incubating the fibers with Fura 2-AM ratiometric fluorochrome (4 μ M) and pluronic acid (0.02%) for 30 minutes at 37 ℃ in culture medium. Fibers were observed with a high resolution digital video camera and intracellular [ Ca ] was estimated by excitation ratio of 340nm/380nm ²⁺ ]。

Figure 2 shows the in vitro effect of compounds AHK1 and AHK2 on quiescent intracellular calcium levels in the mouse muscle fibers. It can be seen how untreated malnutrition fibres (MDX) show a significant increase in calcium levels compared to control fibres (CTRL). Treatment of MDX fibers with AHK1 and AHK2 overnight returned intracellular calcium levels to control levels, demonstrating the ability to restore the intracellular calcium increase observed in muscle fibers. In view of these results, it is assumed that the compounds according to the invention effect this reversion by a mechanism involving the modulation of RyR channels.

Example 26 in vitro assay for RyR 1-Calcilostain 1 interaction

This assay was performed in human control myotubes LHCN-M2 after 9 days in differentiation medium. The myotubes were pretreated with compounds AHK1 and AHK2 at a concentration of 150nM for 12 hours. After treatment, myotubes were subjected to peroxynitrite induced oxidative stress of nitro groups by the addition of SIN1(5mM) for 30 min.

RyR 1-Calcilostain co-localization (colocalization) was analyzed by in situ proximity ligation (in situ PLA) using the Sigma Duolink II network fluorescence kit and RyR 1-and Calcilastatin 1-specific antibodies. This technique allows the detection of precise localisation of two antigens located less than 40nm from each other. To determine RyR 1-Calpain-1 co-localization, quantification was performed using Image J computer software (http:// rsb. info. nih. gov/ij/download. html), 3 pictures in each case, 9 myotubes/field of view (field). The co-localized regions in each image were normalized to the myosin expression region, which was determined by immunofluorescence using fluorescein conjugated specific antibodies.

As shown in fig. 3, the compounds AHK1 and AHK2 according to the invention have the ability to partially restore the reduction of RyR 1-calponin 1 interaction in healthy human myotube cultures after having undergone nitrooxidative stress. Analysis of the RyR1-Calst1 interaction by PLA technique demonstrated: dissociation of the RyR1-Calst1 complex occurs in the presence of SIN1 and can be partially prevented by pretreatment with the compounds AHK1 and AHK 2. The upper panel of figure 3 shows quantification of PLA images by Image J, while the lower panel shows representative images of PLA in each case, with dots representing RyR 1-call 1 interactions (50 μm calibration bar). Thus, the compounds according to the invention tested not only improved the functionality of duchenne-or becker-type dystrophic myotubes, but also minimized the degenerative effects of nitrooxidative stress on healthy human myotubes by increasing the RyR 1-calpain 1 interaction affinity.

According to these results, the compounds of the present invention are useful as therapeutic agents against diseases caused by reduced affinity for RyR 1-calpain 1 under nitrooxidative stress.

Example 27 in vivo biological assays in mice

One month old male dystrophic mdx mice supplied by Jackson laboratories (https:// www.jax.org/strain/001801) were used. Biological assays measuring the effect of ahulken (ahk) compounds on muscle function were performed with one month old mice, while in vivo assays determining the effect on heart and CNS were performed with four month old mice. One month old mice were treated with compound AHK1 or compound AHK2 for 5 weeks, wherein the compounds were administered at a concentration of 0.25mg/mL in drinking water. The muscle strength of the forepaws was measured weekly using a grip dynamometer and the values obtained were normalized by the animal body weight. For this purpose, the instructions described in TREAT-NMD neuro-custom Network protocol (http:// www.treat-NMD. eu/downloads/file/sops/DMD/MDX/DMD _ M.2.2.001.pdf) were followed. Malnutritional mice showed a significant reduction in grip strength. However, after 2 weeks of treatment, the intensity (strength) was significantly increased in mdx mice treated with AHK1 or AHK2 compared to untreated littermate. After 5 weeks of treatment, muscle strength increased significantly by 20% (fig. 4).

This data indicates that compounds according to the invention may be effective in treating patients with muscular dystrophy by improving muscle function or preventing muscle weakness.

After 5 weeks of treatment, the tibialis anterior was obtained and processed for subsequent biochemical and immunohistological analysis to determine the extent of muscle damage. Regeneration by cell death in muscle cryosections was determined by quantifying the percentage of central nuclei using standard immunofluorescence techniques for detecting collagen IV and cell nuclei. Figure 5 shows representative frozen sections of labeled control and dystrophic mouse septa to visualize collagen IV and DAPI for nuclear visualization. Treatment of 5-week AHK1 and AHK2 in mdx mice significantly reduced the percentage of central nuclei, clearly demonstrating the ability of the compounds to reduce histopathological hallmarks of muscular dystrophy after 5 weeks of treatment. These results indicate that the compounds according to the invention are effective in vivo and reach skeletal muscle.

Furthermore, biochemical analysis of tibialis anterior was performed by RNA extraction and expression pattern analysis of control mice, mdx mice and mice undergoing different treatments. For this purpose, a human skeletal muscle, myogenesis and myopathy RT Profiler PCR array (PAHS-099Z, QIAGEN) was used with a cDNA mixture of at least 3 mice/group. The expression profile of 84 genes involved in the pathophysiological mechanisms of skeletal muscle was therefore analyzed. The experiment was performed on a 7300 real-time PCR device (Applied Biosystems) and the results were analyzed with QIAGEN on-line software (http:// www.sabiosciences.com/dataanalysis. php).

Treatment of mdx mice with compound AHK1 for 5 weeks resulted in partial restoration of the gene expression pattern in mdx mice as shown by comparing gene expression in WT and mdx mice in figure 5. Expression of 40 genes was increased in MDX mice relative to controls, and this number was reduced to 20 in mice treated with AHK1(MDX + AHK 1). A gene is considered to be overexpressed when its expression is increased at least 1.75-fold relative to a control. Table 1 shows a list of genes whose expression was increased in mdx mice and decreased to control values by AHK1 treatment, partially decreased by AHK1 treatment or unchanged by AHK1 treatment.

TABLE 1

In particular, the results clearly show that the compounds according to the invention show the ability to reduce the number of genes overexpressed in dystrophic mdx muscle by half. Genes that can be modulated by AHK compounds include, but are not limited to, Akt1, Bcl2, Casp3, Cast, Cav3, Cryab, Ctnnb1, Dag1, Des, Dysf, Foxo3, Igfbp3, Igfbp5, Ikbkb, Mapk3, Myod1, Nfkb1, Ppargc1b, Prkaa1, Rps6kb1, urtr, Casp3, Igf2, Il1b, Il6, Lmna, Mmp9, Myog, Tgfb1, Tnnc1, and Tnnt 1. In this list, Akt1, Il1b, Mapk3, Mmp 9and UtR are genes associated with skeletal muscle loss or atrophy.

To determine the effect of the compounds according to the invention on the heart and CNS, four month old mice were treated with compound AHK2 at a concentration of 0.25mg/mL in drinking water for 5 weeks. The four month old mdx mice showed an exacerbated acute post-stress defense response, which is independent of motor, cardiac and respiratory insufficiency, and is controlled by central mechanisms (fig. 7). Acute stress was induced by manual fixation for 15 seconds in a posture commonly used for performing intraperitoneal injections. Following acute stress, mice were monitored for 1 minute and the percentage of immobility time or tonic immobility time of at least 1 second was calculated, with immobility sensitivity of 90%. No difference was observed in immobility of mice that did not experience stress, indicating that mdx mice showed an exaggerated defense response independent of hypokinesia. Five weeks AHK2 treatment significantly improved the CNS phenotype associated with an aggravated defense response in mdx mice. These results indicate that the compounds according to the invention are effective in treating alterations in brain function in vivo and that they reach the CNS.

The myocardium of four month old, dystrophic mdx mice was highly sensitive to mechanical stress, and the compound isoproterenol induced cardiomyopathy in these mice (figure 8). The integrity of the sarcolemma of the cardiomyocytes was determined by measuring the uptake of evans blue dye accumulated in the membrane-injured cardiomyocytes. Evans blue was administered 24 hours before heart extraction by intraperitoneal injection (10. mu.l/g body weight) at a concentration of 10 mg/ml. Isoproterenol injury was performed by 3 consecutive intraperitoneal injections of β -isoproterenol (350ng/g body weight) 18, 20 and 22 hours after the administration of ivermectin to determine the extent of muscle injury. Regeneration by cell death in muscle cryosections was determined by quantifying the percentage of central nuclei using standard immunofluorescence techniques for detecting collagen IV and cell nuclei. Figure 8 shows representative frozen sections of hearts of control mice and malnutrition mice injected with evans blue to observe cardiac injury caused by isoproterenol. Treatment of mdx mice with 5-week AHK2 increased cardiomyocyte sarcolemma integrity and protected myocardium from isoproterenol-induced injury in mdx mice, indicating that the compound reached the myocardium, was able to modulate srilanca cinnamomi type 2 receptor (RyR2) and was effective in preventing cardiomyopathy in vivo.

Claims

1. A compound of formula (I):

wherein

R ¹ Is C _1-4 An alkylene diradical;

R ² is C _1-4 An alkylene diradical;

R ³ is H;

m is 1;

n is 0;

x is O (C) _1-4 Alkyl) groups; and

y is selected from-CO ₂ H、-N(C _1-4 Alkyl radical) ₂ ，

Or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein R ¹ is-CH ₂ -。

3. The compound of claim 1, wherein R ² is-CH ₂ -or-CH ₂ -CH ₂ -a double free radical.

4. The compound of claim 1, wherein X is meta or para methoxy.

5. The compound of claim 1, wherein Y is selected from CO ₂ H、-NMe ₂ 、-NEt ₂ Or a pharmaceutically acceptable salt of said group.

6. The compound of claim 5, wherein Y is selected from CO ₂ H and-NMe ₂ 。

7. A compound selected from:

1-carboxymethyl-4- [3- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

1-carboxymethyl-4- [4- (methoxy) phenylthiomethyl ] -1H-1,2, 3-triazole,

1-methoxycarbonylmethyl-4- (phenylsulfinylmethyl) -1H-1,2, 3-triazole, and

1-carboxymethyl-4- [3- (methoxy) phenylsulfonylmethyl ] -1H-1,2, 3-triazole,

1-carboxymethyl-4- [3- (chloro) phenylthiomethyl ] -1H-1,2, 3-triazole,

Or a salt thereof.

8. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 7 and one or more pharmaceutically acceptable excipients or carriers.

9. The pharmaceutical composition according to claim 8, for oral or parenteral administration.

10. Use of a compound as defined in any one of claims 1 to 7 for the preparation of a pharmaceutical product intended for the treatment and/or prevention of skeletal muscle disorders and diseases, cardiac disorders and diseases and neurological disorders and diseases.

11. The use according to claim 10, wherein the skeletal muscle disorders and diseases are selected from muscular dystrophy, congenital myopathy, metabolic myopathy, intramuscular atrophy and sarcopenia.

12. The use of claim 11, wherein the skeletal muscle disorder or disease is duchenne muscular dystrophy or becker muscular dystrophy.

13. The use according to claim 10, wherein the cardiac disorders and diseases are selected from heart failure, cardiac ischemia, cardiac arrhythmias and cardiomyopathy.

14. The use according to claim 10, wherein the neurological disorders and diseases are selected from stroke, alzheimer's disease, frontotemporal dementia and cognitive impairment.

15. A process for the synthesis of a compound as defined in any one of claims 1 to 7, comprising:

to produce a compound of formula (I),

wherein:

the radical R in the compounds of the formulae (II) to (III) ¹ 、R ² 、R ³ M, n and X are as defined in any one of claims 1 to 7, and

y is a group as defined in any one of claims 1 to 7, optionally protected with an amino protecting group; and

b) when the compound of formula (I) obtained in step a) has a Y group protected with an amino protecting group, removing the protecting group to yield a compound of formula (I): wherein R is ¹ 、R ² 、R ³ M, n, X and Y are as defined in any one of claims 1 to 7.